Blastomyces dermatitidis septins CDC3, CDC10, and CDC12 impact the morphology of yeast and hyphae, but are not required for the phase transition.

Blastomyces dermatitidis, the etiologic agent of blastomycosis, belongs to a group of thermally dimorphic fungi that change between mold (22°C) and yeast (37°C) in response to temperature. The contribution of structural proteins such as septins to this phase transition in these fungi remains poorly understood. Septins are GTPases that serve as a scaffold for proteins involved with cytokinesis, cell polarity, and cell morphology. In this study, we use a GFP sentinel RNA interference system to investigate the impact of CDC3, CDC10, CDC12, and ASPE on the morphology and phase transition of B. dermatitidis. Targeting CDC3, CDC10, and CDC12 by RNA interference resulted in yeast with aberrant morphology at 37°C with defects in cytokinesis. Downshifting the temperature to 22°C promoted the conversion to the mold phase, but did not abrogate the morphologic defects. CDC3, CDC10, and CDC12 knockdown strains grew as mold with curved, thickened hyphae. Knocking down ASPE transcript did not alter morphology of yeast at 37°C or mold at 22°C. Following an increase in temperature from 22°C to 37°C, all septin knockdown strains were able to revert to yeast. In conclusion, CDC3, CDC10, and CDC12 septin- encoding genes are required for proper morphology of yeast and hyphae, but are dispensable for the phase transition.

Negative image of blastomyces on diff-quik stain.

BACKGROUND: Blastomycosis is caused by a dimorphic fungus that can be difficult to diagnose in certain situations. The disease is sometimes serious and can be deadly. Diagnosis by fungal serology and urinary antigens is not easy to establish and unreliable. Culture is also time-consuming and is not easy to perform. Thus, documentation of such an organism on cytology offers a quick and cost-effective alternative. This report describes for the first time identification of the 'negative image' of Blastomyces budding yeast. CASE: A 79-year-old man presented with a left lung nodule associated with mediastinal and hilar lymphadenopathy. Fine needle aspiration was performed, and a 'negative image' of a yeast with wide base budding was noted on Diff-Quik (DQ)-stained smears. Blastomyces species were confirmed with periodic acid-Schiff fungal stain. Additionally, the fungal capsule contained focally polarizable material on Congo red stain and lacked mucin with mucicarmine stain. CONCLUSION: Blastomyces yeast forms can be easily identified with DQ staining by their 'negative image'. This feature can be utilized as a quick and cost-effective cytological characteristic to further triage these specimens for confirmation. The information can be of great value to clinicians in making appropriate clinical decisions.

Diagnosis of blastomycosis in surgical pathology and cytopathology: correlation with microbiologic culture.

Blastomycosis, a worldwide disease caused by the inhalation of Blastomyces dermatitidis spores, can be diagnosed by microbiologic culture or morphologic identification in tissue or cytologic material. A retrospective review of cases diagnosed as blastomycosis in surgical pathology and cytopathology was undertaken at a University Medical Center to assess the diagnostic value of morphologic methods and their correlation with microbiologic cultures. Surgical pathology/cytology records were reviewed for the period between January 1998 and April 2007 and 53 cases diagnosed as blastomycosis were retrieved: 38 males, 15 females; age 14 to 77 years, median 48. Twenty-nine cases (54.7%) involved lung, 14 (26.4%) soft tissue/bone, 5 (9.4%) skin, 3 (5.6%) other sites, and 2 (3.7%) involved both lung and skin. Forty-six of the 53 patients (87%) had concomitant cultures: 31 (67.4%) were positive for blastomycosis, 11 (23.9%) negative and 4 (8.7%) showed other fungal organisms. A review of microbiology laboratory results for the same period identified a total of 39 patients who were diagnosed with blastomycosis based on isolation of B. dermatitidis. These included 31 cases (79.5%) that were also diagnosed on histology/cytology specimens, 4 (10.25%) that were not submitted to surgical pathology and 4 (10.25%) cases in which pathologic examination failed to identify Blastomyces. This study shows that blastomycosis encountered in surgical/cytopathology can be reliably diagnosed by morphologic examination allowing for prompt treatment. However, microbiologic cultures still play a major role in clinical management of patients suspected of infection because 10.25% were false negative on morphology in our study.

Microecology of Blastomyces dermatitidis: the ammonia hypothesis.

The precise microecology of Blastomyces dermatitidis is unknown, but the fungus has been associated with nitrogenous waste products and rapidly changing environmental conditions. Ammonia accumulates in certain microenvironments, is toxic to most fungi, but may not be identified in processed soil samples. Ammonia tolerance of B. dermatitidis was investigated with two strains recovered in Wisconsin, one from a dog and the other from an environmental source. The samples were grown on phosphate and HEPES buffered agar media supplemented with mineral salts, low (1 g/l) and high (20 g/l) dextrose and increasing amounts of ammonium sulfate, at pH 7-8.2, in gas-impermeable bags at 20 degrees C. Moderate mold growth and sporulation of the strains were observed at low glucose concentration and calculated ammonia concentrations of 4.2 mmol/l when plates were inoculated with either mold or yeast forms. Three recent B. dermatitidis human clinical isolates also exhibited similar growth on this media, and 4/5 strains tolerated ammonia concentrations of 42-62 mmol/l. Growth of virtually all soil fungi from 206 aqueous slurries of fresh and frozen soil from the northern USA and Canada was inhibited at ammonia concentrations of 2.1-4.2 mmol/l. The ability of B. dermatitidis to survive and grow in organic carbon-poor, high ammonia microenvironments may be important to the competitive success of this fungus. These findings may have implications for other dimorphic fungi.

Identification of Histoplasma capsulatum, Blastomyces dermatitidis, and Coccidioides species by repetitive-sequence-based PCR.

The performance of repetitive-sequence-based PCR (rep-PCR) using the DiversiLab system for identification of Coccidioides species, Blastomyces dermatitidis, and Histoplasma capsulatum was assessed by comparing data obtained to colony morphology and microscopic characteristics and to nucleic acid probe results. DNA from cultures of 23 Coccidioides, 24 B. dermatitidis, 24 H. capsulatum, 3 Arthrographis, and 2 Malbranchea isolates was extracted using a microbial DNA isolation kit as recommended by Bacterial Barcodes, Inc. Rep-PCR and probe results agreed for 97.2% of the dimorphic fungi when > or =85% similarity was used as the criterion for identification. Two H. capsulatum isolates were not identified, but no isolates were misidentified. From 43 of those cultures (15 Coccidioides, 14 B. dermatitidis, 14 H. capsulatum, 3 Arthrographis, and 2 Malbranchea), DNA also was extracted using an IDI lysis kit, a simpler method. Rep-PCR and probe results agreed for 97.7% of the dimorphic fungi when a criterion of > or =90% similarity was used for identification. One H. capsulatum isolate could not be identified; no isolates were misidentified. Using > or =85% similarity for identification resulted in one misidentification. These data suggest that the DiversiLab system can be used to identify Coccidioides and B. dermatitidis and, possibly, H. capsulatum isolates.

Global control of dimorphism and virulence in fungi.

Microbial pathogens that normally inhabit our environment can adapt to thrive inside mammalian hosts. There are six dimorphic fungi that cause disease worldwide, which switch from nonpathogenic molds in soil to pathogenic yeast after spores are inhaled and exposed to elevated temperature. Mechanisms that regulate this switch remain obscure. We show that a hybrid histidine kinase senses host signals and triggers the transition from mold to yeast. The kinase also regulates cell-wall integrity, sporulation, and expression of virulence genes in vivo. This global regulator shapes how dimorphic fungal pathogens adapt to the mammalian host, which has broad implications for treating and preventing systemic fungal disease.

Secondary intracerebral blastomycosis with giant yeast forms.

Secondary central nervous system (CNS) blastomycosis is an unusual manifestation of blastomycosis. We report a case of recurrent intracerebral blastomycosis that presented histopathologically with giant yeast-like cells and multinucleation that mimicked Coccidioides immitis. The yeast forms of Blastomyces dermatitidis usually range in size from 8 to 20 microm in diameter. Large or giant yeast forms (20-40 microm) are rare. The four cases previously reported in the literature involving giant yeast cell forms of B. dermatitidis are reviewed here. Intracerebral blastomycosis should be suspected in patients with signs and symptoms of CNS lesions and histories of primary blastomycosis, or treatment with corticosteroids, or comprised immune systems. The diagnosis should be confirmed by culture which presents typical biphasic microbiologic features.

Cutaneous lesions showing giant yeast forms of Blastomyces dermatitidis.

BACKGROUND: The yeast forms of Blastomyces dermatitidis usually range from 8 to 15-20 micro m in diameter. Larger yeast forms have previously been reported only twice in immunosuppressed patients. In both patients these large forms were seen within the lung. CASE REPORT: We present a 14-year-old cardiac transplant patient, who presented 36 days following his transplantation with acute respiratory distress followed a few days later by erythematous cutaneous papules. RESULTS: Biopsy of a skin lesion showed yeast forms, some greater than 40 micro m in diameter, within and surrounding dermal vessels. Cultures later grew Blastomyces dermatitidis. CONCLUSIONS: To our knowledge this is the first reported case of giant forms of Blastomyces dermatitidis within the skin. With increased iatrogenic immunosuppression, we may expect to see more diverse morphologic forms with deep fungal infections.

Blastomyces dermatitidis with large yeast forms.

Yeast forms of Blastomyces dermatitidis typically range from 8 to 20 microm in largest diameter. We report a rare case of primary pulmonary blastomycosis with an unusual morphology, in which we found significant numbers of large yeast forms ranging from 30 to 35 microm in diameter. To our knowledge, this is only the second reported case of giant forms of B dermatitidis. We also review the literature and discuss the possible association of this unusual morphology with immunosuppression in general and glucocorticoid use in particular.

Laryngeal blastomycosis: a commonly missed diagnosis. Report of two cases and review of the literature.

Blastomycosis is a relatively uncommon fungal disease that most commonly affects the lungs. Other organs may be involved, usually secondary to dissemination of the organism. Laryngeal blastomycosis may occur in isolation from active pulmonary disease. The signs, symptoms, clinical features, and pathological findings of laryngeal blastomycosis mimic those of squamous cell carcinoma. Misdiagnosis may result in inappropriate treatment with potential morbidity. Proper understanding of the clinical presentation and familiarity with the histopathologic features of this disease are therefore imperative. In this paper, we report 2 cases of laryngeal blastomycosis, 1 of which was misdiagnosed as squamous cell carcinoma, clinically and microscopically, with consequent radiotherapy and laryngectomy. In the other case, a clinical diagnosis of glottic squamous cell carcinoma was rendered. However, blastomycosis was identified in a biopsy specimen. We also review cases of isolated laryngeal blastomycosis that have been reported in the English-language literature during the last 80 years. A number of those cases were misdiagnosed clinically and microscopically as squamous cell carcinoma.

Cell wall biogenesis of Blastomyces dermatitidis. Evidence for a novel mechanism of cell surface localization of a virulence-associated adhesin via extracellular release and reassociation with cell wall chitin.

Pathogenic yeast of Blastomyces dermatitidis express a surface protein adhesin, WI-1. Due to the crucial role of WI-1 in adherence and disease pathogenesis, we investigated how the protein localizes to the surface of B. dermatitidis. WI-1 released extracellularly by wild-type yeast coated the surfaces of co-cultured knockout yeast within 3 h of incubation, implying that secreted WI-1 provides a pathway for loading the protein onto the yeast cell wall. In radioligand binding assays, purified WI-1 bound saturably, specifically, and with high affinity (K(d) = 8.3 x 10(-9)) to the cell surface of knockout yeast devoid of WI-1. WI-1 added exogenously, in vitro, to knockout yeast was indistinguishable from native cell surface WI-1 by fluorescence staining and restored adhesivity to the knockout yeast in macrophage binding and phagocytosis assays. Analysis of interactions between WI-1 and elements of the yeast cell wall identified chitin as the anchor point for WI-1. This interaction was shown to hinge on the 24-amino acid tandem repeat sequence of WI-1. Efforts to extract surface WI-1 from the yeast demonstrated that it is fastened to the wall by non-covalent interactions and covalent links between cysteine residues. We conclude that the yeast cell surface adhesin WI-1 localizes to the cell wall, in part, through extracellular release followed by high affinity binding back onto exposed chitin fibrils. These findings point to a novel pathway of cell wall biogenesis in yeast and an unanticipated role for chitin in anchoring and displaying a surface adhesin and virulence determinant.

Variable colonial phenotypic expression and comparison to nuclei number in Blastomyces dermatitidis.

We examined colonial phenotypes of five isolates of Blastomyces dermatitidis at 33, 35 and 37 degrees C on four growth media. Three different colony types were identified: yeast, mycelial, and a mixed type consisting of both yeast and mycelia. Each isolate varied in its ability to grow on the different media and at different temperatures, and in the types of colonies it produced in the various temperature-media combinations. Quantification of the number of nuclei per yeast cell by fluorescent staining revealed no correlation between the number of nuclei per cell and the colonial phenotype. These results indicate that the colonial phenotype of B. dermatitidis varies with the isolate as well as with temperature and culture medium, but is not correlated with the number of nuclei per yeast. These findings could provide a start towards typing B. dermatitidis isolates.

Giant forms of Blastomyces dermatitidis in the pulmonary lesions of blastomycosis. Potential confusion with Coccidioides immitis.

Typical yeast-phase cells of Blastomyces dermatitidis have a characteristic appearance in tissue sections. Fungal morphologic variation occurs infrequently in the lesions of blastomycosis, yet it can complicate the differential diagnosis, particularly if fresh tissue is not available for microbiologic culture. The authors report a case of pulmonary blastomycosis, confirmed by culture and direct immunofluorescence, in which some of the yeast-like cells were abnormally large. These giant yeast-like cells exceeded the size range accepted for the tissue forms of B. dermatitidis; therefore, coccidioidomycosis was considered initially in the differential diagnosis. Otherwise characteristic morphologic features of these cells, in particular multinucleation and the production of broad-based blastoconidia, helped resolve the differential diagnosis. The diagnosis can be confirmed by direct immunofluorescence or microbiologic culture.

Mycelial- to yeast-phase transitions of the dimorphic fungi Blastomyces dermatitidis and Paracoccidioides brasiliensis.

The physiological changes that occur during the mycelial- to yeast-phase transitions induced by a temperature shift from 25 to 37 degrees C of cultures of Blastomyces dermatitidis and Paracoccidioides brasiliensis can be divided into three stages. The triggering event is a heat-related insult induced by the temperature shift which results in partial uncoupling of oxidative phosphorylation and declines in cellular ATP levels, respiration rates, and concentrations of electron transport components (stage 1). The cells then enter a stage in which spontaneous respiration ceases (stage 2), and finally, there is a shift into a recovery phase during which transformation to yeast morphology occurs (stage 3). Cysteine is required during stage 2 for the operation of shunt pathways which permit electron transport to bypass blocked portions of the cytochrome system. The mycelial- to yeast-phase transitions of these two fungi are very similar to that of Histoplasma capsulatum. Therefore, these three dimorphic fungal pathogens have evolved parallel mechanisms to adjust to the temperature shifts which induce these mycelial- to yeast-phase transitions.

Cytologic diagnosis of lung cancer. Principles and problems.

This diagnostic seminar discusses the current status of the principles and problems of cytology as it is applied to the diagnosis of lung cancer. This discussion is divided into four major parts. Part I presents a discussion of cytopreparatory techniques and cytology of the lung in the absence of cancer. The cytology of benign proliferations which may mimic cancer is emphasized. The role of cytology in the diagnosis of pulmonary infectious organisms is noted. Part II discusses lung cancer as manifested in specimens of sputum, bronchial washings, and bronchial brushings. Part III presents some data on the validity of cytology with respect to role of specimen number and type in lung cancer diagnosis and cell typing in lung cancer. The continued usefulness and importance of multiple specimens of sputum for lung cancer diagnosis are documented. Part IV presents a brief synopsis of fine needle aspiration biopsy of lung cancer.

Chitin synthetase from the yeast and mycelial phases of Blastomyces dermatitidis.

Chitin synthetase (E.C.2.4.1.16) from mixed membrane fractions of the yeast and mycelial phases of Blastomyces dermatitidis were compared. The behavior of the enzyme from both phases was very similar: N-acetylglucosamine was stimulatory (Km 8.5 mM for yeast and 3.9 mM for mycelium); substrate Michaelis-Menten kinetics were sigmoidal; substrate Km of enzyme from yeast decreased from 3.0 mM at low N-acetylglucosamine (5 mM) levels to 1.4 mM at high (100 mM) levels; substrate Km of enzyme from mycelium was essentially unchanged at 1.4 mM; temperature optimum was 28 degrees C; pH optimum was 7-7.5; Mg+2 optimum was 5-10 mM. The greatest difference was that enzyme from yeast was extracted in a mostly latent form that required trypsin treatment for maximal in vitro activity while enzyme from mycelium was extracted in an active form which was rapidly deactivated by trypsin treatment.

Conversion of Blastomyces dermatitidis to the yeast form at 37 degrees C and 26 degrees C.

A partially defined agar medium, KT, has been developed and compared with brain heart infusion agar for the conversion of Blastomyces dermatitidis to the yeast form. On the KT medium, the mold form converted to a yeast form within 72 h of incubation at 37 degrees C or after 3 weeks at 26 degrees C. A nutritionally dependent dimorphism in B. dermatitidis was observed.

Enhancing effect of murine polymorphonuclear neutrophils (PMN) on the multiplication of Blastomyces dermatitidis in vitro and in vivo.

The purpose of this study was to determine the effect of murine polymorphonuclear neutrophils (PMN) on the multiplication of the fungal pathogen Blastomyces dermatitidis in vitro and in vivo. With a newly devised method, PMN were obtained in adequate numbers (7 X 10(6) per mouse) and purity (92%) for these studies. In 24 h co-cultures PMN, but not lymph node cells (LNC), enhanced the replication of virulent (V) and avirulent (AV) strains of B. dermatitidis 61% and 34%, respectively. In 72 h co-cultures, multiplication was enhanced even more (V, 180%; AV, 140%), when compared to cultures of B. dermatitidis in medium alone. Viability of PMN was not required, because PMN lysates, but not LNC lysates, were effective in enhancing multiplication. Subcutaneous injection of B. dermatitidis mixed with PMN enhanced multiplication by 90% compared to V alone in a subcutaneous abscess model, whereas mixing with LNC did not enhance multiplication. When AV, a strain which does not by itself replicate in vivo, was injected in vivo with PMN it increased 2.75-fold over 4 days. These findings document the in vivo significance of in vitro enhancement of B. dermatitidis replication by PMN, and support the contention that accumulation and death of PMN in B. dermatitidis lesions may exacerbate this infection in its early stages.